The present invention pertains to an apparatus for testing the sterility of fluids, notably antibiotics and other critical fluids, consisting of at least one hermetically sealed test container composed of a plastic material which is preferably transparent, with said container being connected under sterile conditions, by means of flexible connections and a cannula, to another container intended for samples, and/or to other containers for fluids. The top portion of each test container shall consist of a single element possessing the shape of a cup and outfitted with an inlet fitting, an air inlet, and, in the upper section, a built-in air filter. The bottom portion of the test container shall likewise consist of a single element outfitted with an attachment for draining, a mechanical holder for a microporous test filter, and an outlet attachment. The respective brims of both portions of the test container are to be welded together in a pressure-tight and vacuum-tight form, with the test filter being firmly mounted in an undetachable manner between the generally cylindrical lower brim of the top portion of the test container and the generally cylindrical upper brim of the bottom portion.
In medicine and in many other technical fields, maintaining absolutely sterile conditions for the items which are customarily employed and for any fluids which may be used often constitutes an indispensable requirement. This requirement, for example, is especially applicable with respect to solutions intended for perfusions being administered parenterally to patients in a direct form, as well as to instruments which are to be employed in providing perfusions. On account of the fundamental importance of ensuring sterility of instruments and fluids during use, it is necessary to perform sterility tests on a regular basis and to adhere to strict legally defined requirements during these tests (recommendations indicated by the pharmacopeiae of different nations). It has frequently been observed heretofore that, in testing sterility of antibiotics, particular difficulties arise on account of the specific properties of the pharmaceutical substances. Indeed, it has only been possible to overcome these difficulties by adopting highly elaborate precautions during completion of tests, as well as by performing a correspondingly higher number of individual tests.
In accordance with one research method which was widely employed in the past, fluids which were to be tested in terms of sterility were filtered in test containers composed of a transparent plastic material, with multiple sections (Consult Millipore Corporation printed text MRP-4, "Testing Sterility"). The aforementioned test containers possessed a cylindrical upper portion with a removable cap, with fittings for flexible connections and for an air filter cartridge being situated within the front wall. In turn, the bottom portion of each plastic container included a flat, ribbed filter-holder intended to provide support for a microporous membrane, with a fillet being situated upon the outer periphery, so that the bottom end of the top portion could be screwed into position. As a result of screwing the top portion into position, the microporous membrane could be firmly mounted between the holder and an annular shoulder within the top portion, with said shoulder being outfitted with a rubber toroidal joint. When testing for sterility was to be performed, fluids were poured into the top portion of a container under sterile conditions, after removal of the cap, and these fluids would subsequently flow through the microporous membrane when suction was applied. After filtering, the top portion of the container would be unscrewed under sterile conditions, and the center portion of the filter membrane would be removed, so that it could be placed in a bottle containing a culture medium for bacteria, the external ring of the filter being introduced into a bottle containing culture media for yeasts and molds, where incubation would take place at predetermined temperatures. This type of procedure was necessary on account of difficulties in terms of performing effective rinsing of the outer portion of the filter, which tended to retain products such as antibiotics, which inhibit bacterial growth.
This type of testing is relatively expensive, inasmuch as consistently sterile environmental conditions are required during the various phases, and there is a need for various externally performed procedures, such as screwing together the separate portions of the container, removing the filter membrane, cutting out a section of the membrane, and transferring this section to an incubation vessel. Moreover, highly experienced personnel are required. In addition, the individual portions of the test container are separated by joints, and it is possible for air to enter the container through these joints, thereby causing contamination. Furthermore, for extremely rigorous testing of antibiotics, the edges of filter membranes must be hydrophobic, in order to prevent traces of the antibiotic fluid which is being tested from adhering to the outermost edge of the filter, where it would be difficult to remove these traces of fluid by rinsing.
Recently, there has been widespread use of a sterility testing apparatus of the previously cited type (see U.S. Pat. No. 4,036,698, issued July 14, 1977), wherein two or three hermetically sealed test containers composed of a transparent plastic material are connected under sterile conditions to a cannula, by means of welded flexible connections and by means fo a flow divider. The cup-shaped top portion of each test container constitutes a single component, and its front wall contains an inlet fitting which is securely attached to a flexible connecting element, as well as containing an air inlet with a built-in, undetachable air filter. An annular flange is situated upon the bottom edge of the essentially cylindrical wall of the top portion of the test container. By means of this flange, the top portion of the container is firmly welded in a gas-tight and fluid-tight form to an annular flange situated at the top of the bottom portion of the container. A single component produced from a plastic material is also used to form the bottom portion of the container, which essentially comprises a tapered collecting funnel whose top side contains a series of radial ribs situated at angles, so as to furnish a flat holder for the filter membrane. These ribs extend into an outlet fitting situated a the center. A fundamental advantage of this particular testing apparatus in relation to prior types is derived from its constituting a pressure-right and vacuum-tight system which is self-enclosed, as well as being initially sterile. The apparatus consists of several hermetically sealed test containers which are interconnected by means of welded flexible connections, as well as being connected to a cannula by means of a flow divider. Because air can only enter individual containers through the microporous air filters situated within the front walls, it is possible to eliminate practically all errors attributable to secondary contamination when tests for sterility are being performed. Furthermore, this apparatus, for the first time, represents a means of providing pressurized filtration, so that fluids can be filitered by being introduced into containers under excess pressure, instead of by creating a vacuum within the outlet fitting situated at the center. Pressurized filtration renders it possible, for the first time, to achieve considerably more rapid completion of testing procedures, in addition to permitting practically absolute sterility during testing. After filtering of a predetermined quantity of a sample of a test fluid within multiple containers, it is possible to remove deposits and traces of the test fluid by introducing a sterile rinsing fluid into all of the containers simultaneously. Upon completion of rinsing, various culture media are then placed within the respective containers. This procedure can be performed easily, inasmuch as the cannula is inserted within the septum-seal of a vessel containing a culture medium, while conventional shut-off components can be used to seal the flexible connections extending from the flow divider to individual containers, in order to ensure that the culture medium shall only flow into the test container which has been selected for that purpose. Subsequently, incubation is permitted to occur at predetermined temperatures for a specific period of time, within the respective test containers filled with different culture media. A cap is employed for hermetic sealing of the outlet fitting situated at the center, and it is possible to remove a section of the previously sealed inlet connection in order that it may be placed over the air inlet so as to ensure hermetic sealing.
When this type of apparatus is employed to test the sterility of fluids, excellent results can be obtained with numerous types of media, and the necessary experimental procedures for performing tests are extremely simple and rapid. Nevertheless, the previously cited difficulties in terms of testing fluids containing antibiotic substances continue to be present. In fact, filter membranes are tightly fitted between the top and bottom portions of containers, along the outermost edges, with the two portions being joined to one another by welding. Inasmuch as welding is performed along the outermost edge of the membrane, or through the edge, this portion of the membrane is damaged, and small quantities of antibiotics may therefore accumulate within the area of contact between the membrane edge and the two portions of the container, so that it would only be possible to remove these traces by repeated rinsing. When incubation must continue for relatively long periods of time, minute traces of antibiotics may be carried to the center portion of the filter membrane, by means of diffusion, for example. Under these circumstances; bacterial growth would be inhibited, so that test results may be erroneous. As a means of overcoming these basic practical problems, containers with filter membranes which possess a relatively extensive hydrophobic periphery and a hydrophilic center portion have been used for testing antibiotics and similar substances. Nevertheless, production of filter membranes with a disc-like shape is laborious and expensive, because protracted diffusion processes constitute the only means to rendering the periphery hydrophobic. Furthermore, the filtration yield is reduced, even when filtration under pressure is performed, inasmuch as the hydrophobic periphery is practically fluid-tight and is not involved in the process of filtration.
Another disadvantage arising from use of a hydrophobic membrane periphery is attributable to the fact that oxygen can enter the container through the outlet section and can therefore travel by diffusion to the culture medium located above the filter membrane. This circumstance, during tests for the presence of certain types of bacteria, may affect the results which are obtained. During sterility testing for antibiotics and similar products, it is also absolutely essential for the filter membrane to rest upon the ribs of the filter-holder in a completely flat position, without bending. Indeed, bending contributes to retention of fluid, which cannot be easily removed by rinsing. When a pre-cut filter membrane is secured between the two portions of a container by direct sealing or welding techniques, pressure by the melted plastic material against the filter membrane generates internal radial stress which changes the shape of the filter membrane and produces undesirable bending within the membrane, before the edges of the membrane are firmly secured between the top and bottom portions of the container. In practice, loss of shape can be prevented by imparting sufficient stiffness to filter membranes, by adding stiff fibers to the materials used for producing filters. Although published sources indicate that, insofar as filtration techniques may be concerned, it is advantageous to employ biologically inert cellulose ester membranes with a thickness of 150 micrometers, it is not possible from a practical standpoint to use filter membranes of this type without adding fibers, inasmuch as these membranes do not possess sufficient thickness.
As it is possible to observe, the previously cited difficulties, which especially emerge during filtration of fluids containing antibiotic substances or other critical substances, are influenced by the means adopted for mounting or attaching the filter membrane between the two portions of the test container.